P336: A CHEMOTRANSCRIPTOMIC SCREENING IDENTIFIES THE REVERSAL OF GLUCOCORTICOID RESISTANCE IN NOTCH1 MUTATED T-ALL
Authors: L. Pagliaro, L. Moron Dalla Tor, V. Federica, P. Andrei, L. Monica, S. Kleissle, M. Neuenschwander, A. Gherli, E. Cerretani, A. D’Antuono, E. Simoncini, A. Montanaro, G. Roti
Journal: HemaSphere (2022)

Background: Gain-of-function NOTCH1 mutations are the most common genetic abnormality in T-cell Acute Lymphoblastic Leukemia (T-ALL), accounting for 55-60% of the cases. Consequently, modulators of the Notch pathway, such as γ-secretase inhibitors (GSI), would be expected to have clinical efficacy (Rao, Cancer Res 2009). However, their application was limited by an excess of toxicity due to the suppression of wild-type (WT) NOTCH1 proteins in normal tissue (Deangelo, JCO 2006; Doody, Alzherimer’s Res Ther 2015). In the past, we identified the Sarco-Endoplasmic Ca2+ ATPase (SERCA) as a gatekeeper of the oncogenic Notch1 signaling. Thapsigargin (TG), a potent SERCA inhibitor (SI), possesses an anti-NOTCH1-leukemia activity both in vitro and in vivo by preferentially targeting mutated NOTCH1 proteins over WT ones (Roti, Cancer Cell 2013). Aims: Since SIs display a favorable therapeutic index by targeting mutated NOTCH1 proteins, development of new SIs is under preclinical development (Marchesini, Cell Chem Biol 2020). Thus, is important to establish molecular mechanisms portending resistance in order to develop effective therapeutic strategies to overcome or prevent drug resistance. Methods: We established drug-resistant clones (R) from parental ALL/SIL T-ALL cell line using a stepwise increase in treatment dose with TG. We quantified the abundance of differentially expressed (DE) genes (P adj<0.05) in the sensitive or resistant cell line by RNA-seq and identified enriched pathways in the insensitive SI cell line. In parallel, we screened a small molecule library of nearly 2500 bioactive compounds (from the European Chemical Biology Library provided by EU-OPENSCREEN) in ALL/SIL and ALL/SIL R. Compound hits were marked by their ability to inhibit each cell line or both. Confirmatory experiments were completed in multiple T-ALL preclinical models. Results: ALL/SIL R cells displayed a mutation (c.G770T -> p.Gly257Val) in the ATP2A2 gene occurring between the Asp254-Leu260 in the third SERCA2 transmembrane (TM) helix. This variation avoids the efficient TG binding to the catalytic domain, resulting in a diminished inhibitory effect. These cells showed an increase of TG IC50 >150-fold compared to the parental line while remaining partially sensitive to CAD204520, an inhibitor that binds SERCA in a pocket different from the one occupied by TG (Marchesini, Cell Chem Biol 2020). This result suggests that part of the resistance mechanism is transcriptionally mediated and virtually common to several SI. In SI-R cells, transcriptional analysis of 6241 DE genes revealed enrichment in steroids synthesis and response pathways, including cholesterol and lipid metabolism. Consistently, among small molecules targeting the steroid hormone receptors subfamily 3, glucocorticoids (GC) scored among the top hits in ALL/SIL R. While naïve cells were resistant to GC, R cells showed an enhanced sensitivity at low nanomolar concentrations of several GC including dexamethasone, clobetasol and fluticasone. This effect is at least in part mediated by the GC receptors since the R cells displayed an elevated GC receptors protein level, as showed in western blotting and RT-PCR, and the effect on viability were reversed by RU486 co-treatment. Consequently, the association of SI plus GC displayed a synergistic effect in multiple preclinical models, including steroid-resistant cell lines, primary leukemia cells, and PDX models. Summary/Conclusion: These findings suggest that SERCA-Ca2+ modulation mediates GC and steroid signaling and that innovative SI can pharmacologically modulate glucocorticoid resistance in T-ALL.